Due to their particularly high specificity and sensitivity, immunoassays are frequently used for the detection of proteins in serum, plasma, urine or other body fluid samples for medical and diagnostic purposes. Various analytes, such as Thyroid Stimulating Hormone (TSH), Troponin, Prostate Specific Antigen (PSA) or cardiac hormones are detectable with a high degree of sensitivity by sandwich type immunoassays, but for many analytes the requirements for analytical and functional sensitivity are becoming more and more demanding. This is particularly true in the case of TSH because low levels of TSH (around 0.1 xcexcIU/mL) may be caused by different diseases than very low levels (around 0.01 xcexcIU/mL). Thus, different therapeutic approaches might be chosen based on a TSH measurement. This requires that the reported TSH result must not be skewed by an immunoassay""s imprecision at the low end.
In case of TSH, assays are classified in terms of different generations wherein an assay with a total CV of 20% or better at 1-2 xcexcIU/mL is called a 1st generation TSH Assay. The lowest level of analyte an assay is capable of measuring with an imprecision of 20% is called the assay""s functional sensitivity. TSH assays with a functional sensitivity of 0.1 to 0.2 xcexcIU/mL are called 2nd generation and correspondingly assays with a functional sensitivity of 0.01 to 0.02 xcexcIU/mL are called 3rd generation. At this point, 3rd generation immunoassays for TSH are regarded as the state of the art.
An effective and commonly applied method for the detection of analytes such as TSH involves the use of a first antibody specific to one epitope on an analyte molecule and a second antibody directed against another epitope on the analyte and labeled with an enzyme or other label which is able to generate a signal. This will allow the formation of a sandwich complex of the two antibodies and the antigen. The first antibody can be directly attached to a solid support or bound thereto through a hapten label, such as a fluorescein derivative which itself is bound to the solid support through an immuno reactant. The sensitivity of such assays is determined by a variety of factors such as the affinity of the antibodies used and the amount of signal generated by the conjugate with the second antibody. One possibility of increasing the sensitivity of such an assay is using a macromolecular conjugate of the enzyme and the second antibody. The binding of such a conjugate by a single analyte molecule provides a much higher signal compared to a conventionally prepared enzyme conjugate since there are several signal generating enzyme molecules present in the formed immune complex. A difficulty with this approach lies in the discovery that the macromolecular nature of the conjugate may lead to a substantially increased avidity for other, non-analyte components in the sample of body fluid being assayed (e.g. serum), which can cause under-recovery of the target antigen in native patient sera.
Sandwich immunoassays are potentially affected by interfering substances (e.g. heterophilic factors, complement, human-anti-mouse-antibodies) that can bridge the capture and detection antibodies or block one of the antibodies resulting in falsely elevated or depressed results. In the case of the approach of enhancing the sensitivity using highly polymerized read-out conjugates as described above, a falsely depressed result, often called under-recovery, can be observed with many patient samples, but is normally not encountered in buffer solutions containing the purified analyte. The source of the under-recovery noted in such an assay using macromolecular enzyme-antibody conjugates is believed to be caused by the binding of one or several serum components to the conjugate to hereby complicate its interaction with the analyte or reduce the turnover of the assay""s substrates. For a given sample containing the unknown interferents, the degree of the under-recovery appears to be proportional to the level of polymerization of the enzyme-antibody conjugate used for readout.
In U.S. Pat. No. 4,914,040; there is described a phenomena in which interferents in serum such as rheumatoid factors and anti-Fc immunoglobulins such as IgM lead to misrecovery in immunological sandwich assays due to nonspecific binding reactions. This problem can be dealt with by using Fab and F(abxe2x80x2)2 fragments, for one or both of the specific antibodies used in the assay, so that interferents which are directed to the Fc part of IgG lose their point on the specific immune reagents. This reference notes, however, that interferences continue to occur in some human serum immunoassays despite the use of fragments of antibodies in the reagents. The approach described in this patent to combat the interference is to include in the assay a cross-linked immunoaggregate containing an immunocomponent obtained from an animal species different from the species from which the biological fluid is derived at a concentration of from about 0.1 to about 50 xcexcg/mL of test sample which immunocomplex does not bind with the component to be determined. In a preferred embodiment of this system, the immunoaggregate comprises nonspecific IgG cross-linked to a second macromolecule such as a water soluble protein.
The present invention is an improvement to the method for the determination of an analyte in a test sample of a biological fluid which method involves contacting the sample with at least two immunoreactants which specifically bind with the analyte and one of the immunoreactants is labeled with an enzyme wherein the conjugate made up by the second antibody and the enzyme is polymeric or oligomeric. The improvement involves the introduction into the assay of a third conjugate of the enzyme which is used to label the immunoreactant and a different water soluble protein or a non-proteinaceous natural, synthetic or semi-synthetic polymer or oligomer. This third conjugate, the scavenger-conjugate, which has to be polymeric with size-exclusion chromatography suggesting a required molecular mass of more than 5,000 kD for an ALP-IgG polymer. It is added to the assay formulation in order to reduce the interaction of the unknown interferents and the enzyme conjugate made from the second antibody and the enzyme used for readout. The effectiveness of the scavenger conjugate results in the presentation of the incorporated enzyme in a manner similar to its presentation in the conjugate with the second antibody. Therefore an effective competition of the scavenger conjugate with the readout conjugate for potentially interfering substances is accomplished. While a polymer made up by the readout enzyme only shows a limited amount of effectiveness, polymers made from only the second component of an effective scavenger conjugate (e.g. Bovine Serum Albumin (BSA), Bovine Gamma Globulin (BGG) or Keyhole Limpet Hemocyanin (KLH) or of copolymers from the second component of the scavenger with fragments of itself as described in U.S. Pat. No. 4,914,040 are completely ineffective. High molecular weight heteroconjugates made from the enzyme and another macromolecular component were observed to exhibit the highest potency in decreasing the effect of the interferents on the recovery of the assay.
The method of the present invention is particularly suitable for the quantitative detection of analytes which must be determined in minute quantities in order to accurately diagnose a possible disease state.
Thus, while TSH is a natural choice for application of this improved assay technique, other analytes such as troponin, PSA and insulin are also well suited to its use in determining their concentration in a sample of body fluid.
As previously mentioned, the immunoreactant which is enzyme labeled to provide a detectable signal upon reacting with the analyte and being contacted with a substrate for the enzyme is in the form of a polymer or oligomer created by the interaction of the immunoreactant and the enzyme. The formation of the polymer or oligomer is accomplished in the same or related manner as the interference suppressing conjugate which is introduced to the assay system. While oligomers will serve to increase the recovery of analyte, high molecular weight polymers which form colloidal solutions are preferred to ensure maximum interaction with and capture of the analyte and increased signal when contacted with a substrate for the enzyme. Typically, the enzyme will be alkaline phosphatase, but other enzymes such as horse radish peroxidase and glucose oxidase can be used if desired.
The polymeric conjugate of the same enzyme used to label the immunoreactant and a water soluble protein (different than the immunoreactant) or non-proteinaceous natural, synthetic or semi-synthetic polymer or oligomer is prepared by chemical crosslinking using homo or hetero bifunctional or multifunctional cross-linkers or by heat aggregation. The selection of the crosslinker does not depend on the class of the second macromolecule (natural or synthetic) but on the nature of the reactive groups on its surface. The following crosslinkers are all directed against amino groups, but other groups such as carboxylic acids, hydroxyl groups or aldehydes are possible crosslinking sites and would require different crosslinkers. For chemical crosslinking, cross-linkers are generally necessary although so called zero-length crosslinkers can be employed that are not incorporated into the conjugate but are rather employed to activate the proteins. With heat induced crosslinking, no crosslinkers are required. The preparation is conducted in the presence of a cross-linker such as N-Succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), Succinimidyl 4[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC), 2-Iminothiolane (2IT), glutaraldehyde (GA), Bis(sulfo-succinimidyl) suberate (BS3) or combinations thereof. The cross-linkers are normally used in stoichometric excess (5 to 100 fold) and added to a mixture of the proteins to be cross-linked or to separate solutions of the proteins if multi-step coupling procedures are used as in Example III herein.
Exemplary of water soluble proteins which may be used to form the conjugate are serum albumin; particularly bovine serum albumin; an immunoglobulin such as bovine gamma globulin (BGG), goat IgG or any mouse antibody; keyhole limpet hemocyanin; ovalbumin; and casein and reacting them with the enzyme to form a macromolecular conjugate there is provided a suitable material for reducing or eliminating the interference in the type of assay under consideration which causes under recovery of the sought after analyte. While we do not wish to be bound by any particular theory or mechanism as to how the macromolecular conjugate of the present invention interacts with the assay components to reduce or prevent underrecovery of the analyte, it is believed that it competes with the enzyme conjugate incorporating the second antibody for the interferents present in the biological sample using the same mechanisms of interaction, e.g. van der Waals forces, hydrophobic interaction and hydrogen bonds. The polymeric nature of the blocker increases the avidity of its interaction with interferent compounds. In addition to soluble proteins other materials which will carry reactive groups that allow proper functionalization can be used to form the conjugate with the enzyme. Thus, the use of non-proteinaceous natural, synthetic or semi-synthetic polymers or oligomers can be used in place of proteins. Exemplary of such materials are polylysine, polyasparagine and dextranes which will typically have molecular weights in the range of from 3 to 250 K Daltons.
The enzyme containing scavenger conjugate will typically be treated with heat and/or enzyme inhibitors such as silver, lead or copper salts or be exposed to extreme pH conditions to deactivate the enzyme thereby avoiding false positive results emanating from the enzyme in the conjugate. The conjugate is combined with the other assay reagents in sufficient amount to provide a conjugate concentration of from about 50 to 750 xcexcg/mL with a concentration of at least 150 xcexcg/mL being typical.